1. Field of the Invention
The present invention relates generally to the fields of neurobiology, cell biology, and stem cell bioengineering. More particularly, it concerns methods and compositions for culturing of neuronal precursor cells, including large scale methods.
2. Description of the Related Art
Central nervous system disorders such as Parkinson's disease (PD) and Huntington's disease (HD) affect millions of individuals in North America and cost health care systems over $250 billion/year (Kallos et al., 2003). PD and HD are neurodegenerative disorders characterized by the selective and gradual loss of dopamine (DA)-producing neurons (DAergic neurons) and γ-aminobutyric acid (GABA)-producing neurons (GABAergic neurons), respectively (Storch and Schwarz, 2002). Conventional treatment methods for these disorders are focused on the administration of drugs to alleviate symptoms. However, these medical therapies do not replace the lost cells and become ineffective over time.
Previous studies have shown that cell replacement therapy using fetal tissue may be a viable treatment option for PD (Freed et al., 1992; Lindvall et al., 1990; Lindvall et al., 1994; Piccini et al., 1999; Piccini et al., 2000) and HD (Bachoud-Levi et al., 2000; Bachoud-Levi et al., 2006; Bachoud-Levi et al., 2000; Philpott et al., 1997). In the case of PD, two double-blind studies performed by the National Institutes of Health (NIH) found that the transplantation of fetal DA neurons into the brain of PD patients resulted in modest functional benefits but also caused undesirable side effects (Freed et al., 2001; Olanow et al., 2003). However, recently, Mendez et al. (Mendez et al., 2002; Mendez et al., 2005) demonstrated that a new transplantation procedure with a multiple-graft strategy and placement of cells in both the caudate and substantia nigra of the brain benefited PD patients without the side-effects previously reported. In the case of HD, the results of transplantation of fetal striatal tissue into the striatum of HD patients showed that fetal tissue can be isolated for use in the treatment of HD and that neurological implantation of the isolated cells is safe (Bachoud-Levi et al., 2000; Kopyov et al., 1998; Rosser et al., 2002), and the transplantation of these cells may have clinical benefits with respect to motor and cognitive outcomes (Bachoud-Levi et al., 2000; Philpott et al., 1997; Dunnett and Rosser, 2007).
Despite these encouraging results, the lack of fetal tissue availability may ultimately limit the clinical utility of this treatment approach. For example, reversal of symptoms in a single PD patient typically requires transplantation of primary tissue procured from 5-10 fetuses (Storch and Schwarz, 2002; Dunnett and Rosser, 2004). This issue becomes very pronounced when it is considered that there are millions of individuals who could benefit from this type of therapy. For this reason, together with the fact that fetal tissue is mired in ethical controversy, primary fetal cell therapy may not be of widespread clinical utility (Dunnett and Rosser, 2007). If cell replacement strategies are to become a routine therapeutic option for the treatment of neurodegenerative orders, then cell supply becomes a critical issue.
Human neural precursor cells (hNPCs) expanded in culture may represent a viable alternative to primary fetal tissue in clinical cell replacement strategies. Several studies have focused on development of growth medium and expansion of hNPCs in small tissue culture flasks (Carpenter et al., 1999; Storch et al., 2001; Suzuki et al., 2004; Svendsen et al., 1998; Vescovi et al., 1999). The inventors have also successfully modified an existing serum-free cell growth medium (PPRF-m4) that was originally developed for the expansion of murine neural precursor cells (mNPCs) to now support the expansion of fetal hNPCs. hNPCs obtained from different regions of the fetal brain including the forebrain, ventral mesencephalon, brain stem, and spinal cord have been expanded in both static culture and small-scale suspension bioreactors using this modified medium (PPRF-h2). hNPCs expanded in standard small-scale suspension bioreactors have been effectively used for transplantation studies in animal models of PD, HD, and neuropathic pain (Mukhida et al., 2007; Mukhida et al., 2006). The results showed that hNPCs expanded in small-scale bioreactors could be differentiated into a GABAergic phenotype which, when transplanted into lesioned animal models of neuropathic pain (Mukhida et al., 2007) and HD, resulted in significant functional recovery. Moreover, when these cells were co-grafted with fetal VM derived tissues, they enhanced the fetal dopaminergic neurons' survival following transplantation into a rat model of PD (Mukhida et al., 2006), suggesting a possible neurotrophic role for the bioreactor-expanded hNPCs.
In order for such cells to be approved for use in clinical settings, they must be generated in a reproducible manner under controlled, standard conditions. Small volume bioreactor methods make it difficult to incorporate the measuring probes necessary to monitor and properly control environmental parameters of the cell culture, and also do not permit the efficient production of sufficient cells to allow small quantities of primary hNPCs, isolated from a single fetus, to be expanded to clinical quantities. Thus, improved methods and compositions for large scale culturing of NPCs are needed.